Tobiano is a white spotting pattern in horses caused by a dominant gene, Tobiano (TO). The tobiano color pattern of horses is highly valued and selected by many horse owners and breeders. Approximately 350,000 horses carrying TO are currently registered by the American Paint Horse Registry (APHA), but the pattern can also be found among several different horse, pony and draft breeds worldwide.
TO was previously shown to be genetically linked to the gene for Albumin(AL) (Trommershausen-Smith 1978). Later, an allelic association (linkage disequilibrium or LD) was discovered in which the chromosome possessing the TO allele usually possessed the Albumin (AL)-B and Vitamin D binding factor (GC)-S alleles (Bowling 1987). It is known that certain chromosome rearrangements, such as inversions, can create unusually strong LD by interfering with gamete formation, thus resulting in the formation of conserved haplotype blocks of alleles.
To explain the unusually high level of LD between the TO, AL and GC loci it was hypothesized that an inversion on ECA3 could be preventing recombination in this region (Bowling 1987). Several similar spotting patterns at the W locus in the mouse have been shown to be due to chromosomal rearrangements near the KIT (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncongene homolog) gene. Two inversions, Rump-white (Stephenson et al., 1994) and Sash (Nagle et al., 1995), and four deletions, Patch, 19h, 57, and Banded (Nagle et al., 1994, Kluppel et al., 1997) occur within the 200 kb upstream of the KIT gene. All have been shown to disrupt the tissue-specificity or temporal expression of KIT during embryogenesis (Nagle et al., 1994; Kluppel et al., 1997; Hough et al., 1998; Berrozpe et al., 1999) rather than a coding sequence. The similarities between Tobiano and this particular group of mouse spotting patterns, previously demonstrated linkage of Tobiano and KIT horse chromosome 3 (ECA3) (Brooks et al., 2002) and the lack of a difference in the KIT cDNA of Tobiano horses (Brooks, 2006) all suggested that a nearby chromosomal rearrangement was the cause of the Tobiano allele. Initial efforts to detect an inversion using cytogenetic methods were unsuccessful (Raudsepp et al., 1999).
The rearrangement was finally demonstrated by one of the present inventors (Brooks, 2006; Brooks et al., 2007). Cytogenetic evidence showed that there was, indeed, a chromosome inversion on ECA3 near KIT which appeared to be completely associated with TO. No exceptions to the association of Tobiano with the inversion have been found and it is likely that the inversion has this effect by affecting regulation of the gene, KIT, during embryonic development. This conclusion is also based on observation of similar effects of mutation in KIT on mouse hair color.
The results of mapping several genes are shown in FIG. 1 and Table 1. In the left hand column of FIG. 1, a clear difference can be seen in the distance between KDR (red) and KIT (orange) when comparing the ECA3 from non-spotted horses and the ECA3 bearing TO. The center set of images shows three markers (ALB, Clock, and GABPB1) with different relative orders on each chromosome. The right column shows results from FISH mapping a bacterial artificial chromosome (BAC) 558 which crosses the distal breakpoint (green). On ECA3 from non-spotted horses, hybridization occurred at a single location, while on the chromosome with Tobiano two distinct locations were labeled with the single probe.
TABLE 1FISH Markers used in this study in their relative linear order on ECA3.CHORI-241 BACSequenceMarkerClonesHSAECARelation to InversionSourceGABRB149:M134q046.8mb3q21TelomericUCSC ConsensusTEC38:G14q047.9mb3q21TelomericUCSC ConsensusPDGFRA23:F114qos4.93q21TelomericUCSC ConsensusKIT e2190:F84q055.447mb3q21TelomericGenbank#AY874S43Intergenic Seq.102:M14q055.558mb3q21At BreakpointUCSC Consensus“558”KDR5′Ue1129:044q055.787mb3q21WithinUCSC ConsensusKDR127:D234q055.7mb3q21WithinUCSC ConsensusClock11:A94q056.2mb3q21WithinMurphy et al.2007ALB21:KS4q074.6mb3q14.WithinGenbank#AY008769CCNI99:894q078.4mb3q14.WithinChowdhary et al.2003ENOPH169:C104q083.7mb3q13WithinGenbank#cxs03024WDFY344:L244q085.9mb3q13WithinGenbank#CX599384HSD17B11105:8194q088.7mb3q13WithinChowdhary et al.2003PDLIM519:G114q095.7mb3q13WithinUCSC ConsensusADH1C189:L204q100.6mb3q13CentromericGenbank#AF134056
Since the presence of the tobiano pattern can increase value, horse breeders prefer to use breeding stock which are homozygous for TO, e.g., those which have inherited a copy of the gene from both parents, and will always transmit the gene to their offspring. The cytogenetics test requires freshly collected cells and is expensive to conduct. In place of a cytogenetic test for Tobiano, associative tests have been used to predict homozygosity for Tobiano. As described above, the Tobiano gene was associated in horse populations with particular gene-alleles or SNPs. Specifically, most but not all horses with the Tobiano gene also possessed the B allele for AL and the S allele for GC. Likewise, most but not all horses with the Tobiano gene also possessed the KM1 single nucleotide polymorphism of the gene KIT. Known methods for detecting genetic markers associated with the Tobiana genotype include detection of these nearby genetic markers using biochemical typing or molecular DNA tests. However, biochemical typing methods require freshly obtained biological samples. Furthermore, both biochemical and molecular gene detection methods also produced false positive and false negative reactions because they are associative with the tobiano trait and not actually responsible for the trait (Duffield and Goldie, 1998; Brooks et al., 2002).
Until present, there have been no precise and cost effective methods to identify all horses homozygous for TO. While the inversion can be detected using cytogenetic studies and fluorescence in situ hybridization (FISH) of DNA probes for genes in the region, this is an expensive and time-consuming procedure. There remains a need in the art for a methods for identifying animals bearing the Tobiano genotype which are effective, rapid, and which further can be performed effectively on stored samples of varying type, to obviate the need for acquiring fresh genetic material.